Donkey whey protein and peptides regulate gut microbiota community and physiological functions of D‐galactose‐induced aging mice

Abstract The prolongation of life span has attracted more and more attention in the current world. Gut microbiota is considered one of the most critical elements and is essential in regulating life span and quality. The effects of donkey whey protein (DWP) and donkey whey hydrolysate (DWPP) on physiological functions and gut microbiota of D‐galactose‐induced aging mice were investigated to find new strategies for resisting aging. Our results showed that DWP and DWPP could increase the body weight gain velocity, superoxide dismutase (SOD) activity, and thymus index, whereas decrease the level of reactive oxygen species (ROS) and malondialdehyde (MDA), and improve the aging of the body in the liver congestion, oozy draw focal sclerosis of chronic inflammation. The effects of medium and high concentrations of DWP and low and medium concentrations of DWPP were the same as the vitamin C (Vc)‐positive control group. It was found that both DWP and DWPP could change α‐diversity; the relative abundance of Lactobacillus increased, whereas the relative abundance of Helicobacter and Stenotrophomonas decreased after being treated with DWP and DWPP. The correlation between intestinal microflora and physiological indexes showed that chao1, ACE, and observed species indexes in the α index were positively correlated with weight gain velocity, SOD activity, and thymus index. The relative abundance of Lactobacillus was positively correlated with SOD and thymus index but negatively correlated with MDA. The relative abundance of Stenotrophomonas was opposite to that of Lactobacillus. The Anaerobiospirillum, Fusobacterium, and Dubosiella had a significant positive correlation with the weight gain velocity. The study provided a deeper more profound understanding of the potential use of DWP and DWPP in senescence delays.


| INTRODUC TI ON
The prolongation of life span has attracted more and more attention due to the increase in economy and life quality. However, it is still a global challenge to improve the health span of people (Dzau et al., 2020;Partridge et al., 2018). Aging, which is the timedependent functional decline affecting most living organisms, has attracted curiosity and excited imagination throughout the history of humankind (López-Otín et al., 2013). Aging is a complicated process that is influenced by a variety of factors such as the gastrointestinal/digestive system, immune system, nervous system, metabolic system, various biological signaling pathways, programmed cell death (PCD), and environmental factors (Adriansjach et al., 2020). A better understanding of aging is necessary for developing novel approaches to prolong the life span and increase life quality, especially for the elders.
Among the factors affecting aging, the gut microbiota are considered one of the most critical elements and plays an essential role in regulating life span and quality (O'Toole & Jeffery, 2015). The microbiota composition of the gut changes with age, and alterations in this composition affect human health (Odamaki et al., 2016;Xu et al., 2019). A large body of microorganisms resided in the gut, approximately between 10 13 and 10 14 (Liu et al., 2020). The composition of gut microbiota has been shaped in early childhood, becomes mature by the age of 3 years, and stays stable over the life span (Fouhy et al., 2019). The 16S ribosomal DNA-sequencing data of fecal samples indicated that the structure and diversity of gut microbiota got remarkable changes in older adults compared with young settings (Kim & Jazwinski, 2018). It was found that the proportion of bacteroides, clostridia, and total lactobacilli declined, while the population of fusobacteria, streptococci, and staphylococci increased in older adults compared with the younger group (Woodmansey et al., 2004). The changes in structure and diversity of gut microbiota often influence innate immunity's homeostasis, further affecting body functions and health (O'Toole & Jeffery, 2015). Herein, the investigations toward the relationship between gut microbiota and aging will open up new opportunities for discovering new strategies to slow down aging. Donkey milk has been recognized for nutritional and therapeutic purposes since ancient times (Cunsolo et al., 2017). Donkey milk is an excellent replacer of cow milk for infants who suffer from cow milk protein allergy (CMPA), and its composition (e.g., the contents of both lactose and protein) is quite similar to human milk (Martini et al., 2018;Souroullas et al., 2018). Emerging evidence shows that donkey milk has certain physiological functions, including high tolerability, antimicrobial, and anticancer activities .
However, the effects of donkey milk on gut microbiota remain unclear and need further investigation.
Therefore, the present work aimed to investigate the effects of DWP and DWPP on the composition and abundance of gut microbiota in D-galactose-induced aging mice. The microbial community and relative abundance were evaluated by 16S rRNA gene sequencing, and the relationship between gut microbiota and physiological and functional features was predicted by correlation analysis. The results are expected to improve our understanding of the association between gut microbiota changes and aging, which may provide practical information for discovering the underlying mechanism of aging and novel approaches to improving aging problems.

| Materials
Donkey milk was obtained from the local farm (Lanzhou, China), taken to the laboratory within 1 h, and stored at −20°C for further use. All other chemicals and reagents used in this work were provided locally and were of analytical grade.

| Preparation of DWP and DWPP
For the DWP preparation, donkey milk was centrifuged with a centrifuge (Heraeus Multigauge X1R, Thermo Fisher) at 4700 g (4°C) for 30 min. The upper fraction (milk fat) and a down fraction (milk whey and granule) could be visualized after centrifugation. Upper milk fat was discarded, and the pH of left skim milk was adjusted to 4.6 with 1 M HCl, followed by incubating in the water bath (40°C) for 20 min.
Then, skim milk was centrifuged at 12,000 g at 4°C for 20 min, and supper milk whey (MW) was collected and then lyophilized. The MW powder was stored at 4°C until further use.
For DWPP preparation, 0.5 g DWP was dissolved in pure water and the final concentration was adjusted to 30 mg/ml. Then, it was incubated at 90°C for 5 min, followed by cooling down to 50°C by stirring with a magnetic stirrer, and adjusted pH to 7.0. The concentration of neutral protease was carefully adjusted to 4000 U/g. Hydrolysis lasted for 5 h; during the hydrolysis, pH was kept to 7.0. After hydrolysis, the reaction system was heated to 100°C for 10 min to inactivate the protease. Then, the reaction system was centrifuged at 15,000 g for 10 min, and the upper fraction (DWPP) was collected, then lyophilized and stored in the fridge (4°C) for the following experiments.

| Chemical composition of DWP and DWPP
The Kjeldahl method determined the protein content (AOAC, 1995).
The conversion factor used to convert the Kjeldahl nitrogen value (N) to protein amount was 6.25. Results were expressed as g per 100 g dry weight (g/100 g DW). Fat and lactose content were determined by the method described in GB5009.6-2016 (National Standard of the People's Republic of China: national standard for food safety, determination of fat in food, China, 2016) and GB25595-2018 (National Standard of the People's Republic of China: national standard for food safety, determination of lactose in food, China, 2018), respectively. The pH-stat method was used to examine the hydrolysis efficiency of DWP.

| Animal experiments
All (total of 65) healthy female Kunming mice (age 6 weeks, 25-30 g body weight) were provided by the Experimental Animal Center of the Institute of Genetics and Developmental Biology of the Chinese Academy of Sciences (Lanzhou, China). The mice were kept under standard laboratory conditions and housed in an environmentally controlled room (24 ± 1°C) with 55 ± 10% relative humidity in a 12 h light/dark automatic lighting cycle. They could access the standard pellet diet and drink water ad libitum throughout the study. The  Table 1. Physiological criteria, including body and thymus weight, were determined following the methods described by Farhangi et al. (2017) and Kong et al. (2018). The weight gain velocity (Sié et al., 2021;Stahr et al., 2021) was calculated with the formula below: when: N > 2, M = N-2; N = 1, M = 0, Next-generation sequencing (NGS) of the 16S rRNA gene amplicons method was utilized to check the gut microbiota in the intestinal contents collected from three mice (they were selected from every group randomly).

| Assays of SOD and MDA
The superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were determined with the commercial kit manufactured by Nanjing Jiangcheng Bioengineering Institute (Nanjing, China) according to the instructions.
The sample and reagents were added to the tubes and incubated at 37°C for 40 min. And then, a chromogenic reagent was added and mixed. After being placed at room temperature for 10 min, the absorbance was recorded by a Microplate Reader (Bio-Rad 550). As for MDA content, the sample and reagents were added into the tubes and incubated at 95°C for 40 min. After cooling to room temperature, they were centrifuged at 1360 g for 10 min, and the absorbance of the sample was measured with a Microplate Reader (Dai et al., 2008).

| ROS lever determination
ROS level was determined according to the method described in the previous study (Zhong et al., 2019). One hundred milligram of liver tissue was mixed with 1 ml buffer and homogenated using a grinder.
Then, the mixture was centrifuged at 4°C for 10 min and 190 μl of supernatant was incubated with 10 μl of BBoxiProbe O13 ROS probe (BestBio) in a 96-well plate at 37°C in the dark for 25 min. ROS levels were quantified by a fluorescence microplate reader (Bio-Rad 550) weight gain velocity = weight at N weeks − weight at M weeks weight at M weeks × 100 % . with an excitation wavelength of 488 nm and an emission wavelength of 606 nm.

| Histological assessment of liver
Hematoxylin and eosin (H&E) staining of the liver was performed according to the method described in the previous study (Kong et al., 2018). Liver tissue was collected and fixed in 4% paraformaldehyde (v/v) for 24 h. Then, samples were dehydrated in ascending grades of alcohol, then embedded in paraffin, and finally, sectioned at a thickness of 5 μm. H&E stained these samples for routine examination of these tissues, and pictures were recorded by light microscope (Olympus CX31). Bioanalyzer (Agilent) were used to determine the densities of the collected fragments and execute quality control, respectively. The efficiency of the adapters was evaluated by qPCR. The sequencing process was accomplished using the IonS5TMXL system (Thermo Fisher Scientific).

| Sequence data analysis
The raw data were filtered using Cutadapt (V1.9.1) quality-controlled process to remove barcode, sequences of 5′-end primer mismatch base number >1, sequences with length ≤150 bp, sequences with a continuous equal number of bases >8, and ambiguous sequences.
Then, the reads were submitted to the Silva database (https://www.

| Chemical composition of DWP and DWPP
The chemical composition and hydrolysis efficiency were determined and results are shown. The protein content was 10.4 ± 0.6 g/100 g, lactose content was 78.9 ± 0.8 g/100 g, and fat content was 0.396 ± 0.14 g/100 g. The hydrolysis efficiency was 10.2%.

| Effects of DWP and DWPP on the physiology of D-galactose-induced aging in mice
It is attractive to investigate the effects of DWP and DWPP on the physiology of D-galactose-induced aging mice, since donkey milk has been demonstrated to play an essential role in maintaining body functions. As shown in Figure 1, at the end of the 3rd, 5th, and 7th weeks, the weight gain velocity of the intervention groups except DWPPH group was higher than that of the aging group, and the changes in DWPH, DWPPL, and DWPPM groups were similar to or even better than that of the Vc group (DWPPM groups with Vc groups, p < .05; and (e) DWP and DWPP intervention in aging mice liver tissue morphology. In the picture, "→" is the liver tissue with high coagulation necrosis and cirrhosis. "←" is the serous exudate gathered in the hepatic sinusoid space. "↓" is necrotic or apoptotic liver cells. It can be seen that the nuclei of the liver cells are fragmented, the nuclear membrane disappears, and the nucleolus is dissolved, and some nuclei shrink. NS: nontreatment control; Aging: negative control; Vc: positive control; DWPL, DWPM, and DWPH represent low-, middle-, and high-concentration DWP intervention group, respectively; DWPPL, DWPPM, and DWPPH represent low-, middle-, and high-concentration DWPP intervention group, respectively. Histograms on top of which the same letter appears to represent means that are not statistically different; different letters identify means that are significantly different (p < .05).
indicating that donkey whey powder and its hydrolysates showed promising antioxidant activity in D-galactose aging mice. The thymus index in all the intervention groups was significantly higher than the aging group, but not from the NS and Vc groups, indicating that donkey whey powder and its hydrolysates can significantly improve the immune function of D-galactose aging mice (Figure 1d). In contrast, the hydrolysates of donkey whey powder with high concentration showed slight improvement, and there was no statistical difference between the aging groups.
As shown in Figure 1e,  gions, which might be organized nodules produced after liver injury.
In the DWPPH group, atrophy of the hepatic cord and dilatation of hepatic sinusoidal space were also observed, during which a large amount of serous exudation was clustered and distributed, which might be due to the liver edema further causing the compression of hepatocytes in the liver.

| Variations in richness and diversity of the microbial community
Firstly, the raw data were filtered and nonspecific amplified fragments and chimeras were removed; thus, quality control data were obtained (Table S1). It showed that the average clean reads obtained from each group ranged from 65,618 to 82,591. A rarefaction curve was constructed through random sampling of clean reads, which indicated that the curve tended to be flat (Figure 2a). It means that there are enough reads being included in the study. Principal component analysis (PCA) showed differences in the bacterial compositions of the nine groups ( Figure 2b). These results showed that the DWPPL, normal, and Vc intervention groups were far away from the aging group, indicating that the intestinal flora richness and diversity were significantly different (p < .05).
Meanwhile, the results of the alpha-diversity analysis showed that the richness of the microbial community in aging is lowest by comparison with other groups according to alpha-diversity indexes, including Shannon index, Simpson index, chao1, and ace. For DWPand DWPP-treated groups, except DWPH and DWPPH, other groups had increased Shannon value, Simpson value, chao1 value, and ace value compared with aging ( Figure 3); the results of DWPPM were closer to the Vc group. These results showed that besides the high-concentration groups, the donkey whey and its hydrolysates significantly improved the intestinal flora in the senile state, especially DWPPM.

| Effects of DWP and DWPP on gut microbiota community
Based on relative abundance data, Lactobacillus, Helicobacter, and Stenotrophomonas were the most abundant in all groups by comparison with other bacteria (Figure 4a). Lactobacillus in the NS group, Vc, and all the donkey whey and its hydrolysates intervention groups was significantly higher than that in the aging group (p < .05; Figure 4b), followed by Stenotrophomonas, whose relative abundance in the aging group was significantly higher than that in the other groups (p < .05; Figure 4c). There was no significant difference in the relative abundance of Helicobacter between groups. But aging was significantly higher than in the other groups, especially in the NS group, Vc group, and DWPH group (p = .143, .129, and .159, respectively) (Figure 4d). These results showed that DWP and DWPP have effects on the relative abundance of intestinal bacteria, and the detailed mechanism needs further investigation.
F I G U R E 2 Variations in richness and diversity of the gut microbial community. (a) The rarefaction curve was constructed through random sampling of clean reads. (b) Principal coordinates analysis (PCoA) of beta diversity in the microbial communities in each group.

| Correlation between alpha diversity and physiological characterizations
Exploring the relationship between alpha diversity of gut micro- The chemical composition analysis shows that the main components of whey powder are lactose and whey protein. Lactose is hydrolyzed into glucose and galactose, the former mainly provides energy for the body, while galactose binds to ceramide with a glycosidic bond to form galactose cerebral glycoside, which is involved in brain development (Sinha et al., 2007). It was found that the nutrition value of whey protein was much higher than other proteins (Sinha et al., 2007) as it contains rich essential amino acids and branchedchain amino acids, as well as a balanced source of sulfur-containing amino acids including isoleucine, leucine, and valine (Almeida et al., 2016). These essential amino acids play an important role in weight control, obesity prevention, various metabolic functions, and blood glucose homeostasis (Teixeira et al., 2019). In our study, the supplantation of DWP and DWPP with different concentrations can recover weight loss in the aging process, which was consistent with the nutritional supplement of lactose and whey protein.
Aging is accompanied by oxidative damage and changes in immune function. SOD is an important enzyme-exerting antioxidative functions, and MDA is negatively correlated with antioxidative functions (Rasmussen & Suomi, 1989). The SOD activity improved, whereas the MDA content and ROS level decreased with the treatment of DWP and DWPP (Figure 1b,c), indicating

F I G U R E 3
The alpha-diversity analysis regarding microbial communities. The richness of the microbial community in aging is lowest compared with other groups according to alpha diversity indexes including Shannon index, Simpson index, chao1, and ace. Histograms on top of which the same letter appears represent means that are not statistically different; different letters identify means that are significantly different (p < .05). NS: nontreatment control; Aging: negative control; Vc: positive control; DWPL, DWPM, and DWPH represent low-, middle-, and high-concentration DWP intervention group, respectively. DWPPL, DWPPM, and DWPPH represent low-, middle-, and high-concentration DWPP intervention group, respectively.
that DWP and DWPP exhibited good antioxidant activity. Lactose has a terminal aldehyde group (HC=O) and can be oxidized to COO- (Considine & Frankish, 2013). Five millimeter lactose can not only reduce free radicals to 53.5% but it also plays a particular role in the formation of peroxide free radicals (Wehmeier & Mooradian, 1994). Whey protein is a complete protein rich in sulfur-containing amino acids (Rastall et al., 2005) and branched amino acids, which can be used as a precursor of the antioxi- Nevertheless, many findings indicate that high protein intakes (more than 40% protein level) increase oxidative stress (Toyomizu et al., 1995) and decrease antioxidative activity in mice's digestive organs. This imbalance between the production of free radicals and the ability of the organism's natural protective mechanisms is due to the excessive oxidation of amino acids (Gu et al., 2008). Therefore, DWPPH could increase the thermogenic response, which is accom- The gut microbiota is crucial in regulating energy homeostasis and intake to control body functions Yin et al., 2019). It was also demonstrated to be closely linked to aging (Adriansjach et al., 2020). Whey protein is essential in maintaining gut development and functions (Hahn et al., 2018). Firstly, lactose promotes gut health because the microbiota use it as a carbon source. Its fermentation is followed by carboxylic acids (lactic and butyric) that decrease the gut pH and create a nonfavorable medium for pathogenic bacteria (Kareb & Aïder, 2019). Secondly, whey protein also acts as a prebiotic in the intestinal tract, increasing the abundance of Lactobacillus in mice (Boscaini et al., 2020;Sánchez-Moya et al., 2017). In humans, it was found that whey protein could enhance the diversity of gut microbiota to improve weight loss and/ or alter appetite (Reimer et al., 2017). Different from cow milk, donkey milk was found to be closer to the composition of human milk, which makes it exert a particular function, such as improving life quality for infants allergic to cow milk (Sarti et al., 2019). Therefore, this experiment was conducted to study the effects of donkey whey powder and its hydrolysates on the intestinal microflora of aging mice, and to provide a theoretical basis for supplementing donkey whey powder in the aging population and slowing down aging.
High diversity of intestinal flora is associated with health, low diversity is associated with different diseases, and community diversity is negatively correlated with the vulnerability of the elderly species were positive correlated with the weight gain velocity, SOD, and thymus index. Therefore, appropriately increasing the diversity of intestinal flora can promote the body to improve antioxidant capacity and resist aging. But more needs to be done to support this mechanism (Ma et al., 2021).
The structure of intestinal flora is affected by many factors, such as country, region, diet habits, gender, and disease, but it is still considered to be one of the crucial factors in determining longevity (Han et al., 2017;Santoro et al., 2018) and has been found that the gut microbiota structure of the elderly is very different from that of the young (Gruber & Kennedy, 2017;Park et al., 2015).
In the elderly subjects, the abundance of Firmicutes decreased and the abundance of Bacteroidetes increased (Kumar et al., 2016),

| CON CLUS ION
The present work explored the effects of donkey whey protein and peptides on the composition and abundance of gut microbiota in D-galactose-induced aging mice. The result showed that DWP and DWPP could change the alpha diversity and the relative abundance of gut microbiota in D-galactose-induced aging mice. DWP and DWPP can improve antioxidative activity. DWP and DWPP increase the relative abundance of Lactobacillus and decrease the relative abundance of Stenotrophomonas and Helicobacter.
The concentration remarkably influences the effects of DWP and DWPP on gut microbiota and physiological functions in aging mice.
The study highlights the importance of DWP and DWPP in improving the health status of aging settings and opens new opportunities for future advances in developing novel strategies that slow down aging.

CO N FLI C T O F I NTE R E S T
The authors have no conflicts of interest to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data will be made available on request.